Performance of Fungal Growth Through Integrated Gompertz Model and Respiratory Quotient by Solid State Fermentation in Multi-Layer Squared Tray Solid State Bioreactor with Aeration Strategies

A newly designed, laboratory-scaled and multi-layer squared tray solid state bioreactor (SSB), was developed and successfully operated in solid state fermentation (SSF) conditions. The bioreactor was divided into eight layers of squared perforated trays. Wheat bran was used as a solid substrate for the growth of Aspergillus awamori and Aspergillus oryzae . The SSB was equipped with an oxygen (O 2 )/carbon dioxide (CO 2 ) gas analyser and a thermocouple. Continuous on-line monitoring of fungal growth could be performed by indirect methods that measure O 2 consumed, production of CO 2 and metabolic heat. The advantage of using this method is that there are no tedious and time-consuming sampling processes. The evolution of CO 2 , which represents an accumulation term, was integrated with time and fitted to the Gompertz model in a log-like equation. The Gompertz model generated values that may be used to stimulate and verify the experimental data. Results strongly suggest that the evolved and accumulated CO 2 , excellently described fungal growth. Simulated results agreed with experimental results. The respiratory quotient (RQ), which is the ratio of CO 2 evolution rate (CER) to O 2 uptake rate (OUR), was determined by the gas balance method. CER and OUR confirmed that measurements correlated to fungal activity. Each RQ values can explain the differences of each SFF process carried out. Yet, heat evolved by fungal activity also described fungal growth. The current findings is an excellent pre-liminary experimental work, evidencing that multi-layer squared tray SSB with forced moistened aeration present a promising alternative of instrumented bioreactors for SSF processes.


Introduction
Solid state fermentation (SSF) has shown much promise in the development of several bioprocesses and products, with high volumetric productivity, relatively higher concentration of the products, less effluent generation, and simple fermentation equipment or bioreactor systems. Since 2000, SSF has received significant attention for the expansion of industrial bioprocesses, mainly due to its lower energy requirements and association with higher product yields, lower catabolic repression, costeffective technology and less wastewater production with a lower risk of bacterial contamination [1].
In addition, SSF can be identified as an environmentally friendly process, as it uses solid residues from agriculture industries as the source of carbon substrate.
Along with the development and modernization of technology, SSF will soon be related to a solid state bioreactor (SSB) design which is a vital development and advancement in the bioprocessing technology.
In any aerobic SSF processes within any type of SSB, there are a few identified factors in determining the performance of the fermentation process. They are substrates heterogeneity, water activity or moisture gradients, O2 supply, carbon dioxide (CO2) transfer, temperature, metabolic heat removal and the bioreactor's design itself. Operational difficulties include issues related to poor O2 supply, moisture level, heat accumulation in the bioreactor system and controls, which needs to be highlighted [33].
During the fermentation process, heat is generated and can consequently and directly affect the metabolic activities of the microorganism, causing, amongst others, moisture loss. Moreover, if the temperature control and aeration fail, the selectivity of the biosynthesis process towards product formation could be lost. Additionally, there is potential for the process to fail completely. A good strategy is therefore needed to effectively remove heat from the system [34]. Moisture content, heat and evolvement of CO2 are requirements strongly linked to O2 transfer rates in aerobic processes [35][36][37].
Direct aeration is the principal operation that results in O2 transfer, moisture loss, heat and CO2 removal.
However, these effects are increased when high biomass concentrations and microorganisms (particularly, filamentous fungi) are fed into the SSF, creating a complication with its use. A strategy to control these effects is to introduce forced aeration. Forced aeration allows on-line analysis of CO2 and O2 concentrations in the exhaust gas that is in accordance with and controls the fermentation process [34]. In addition, analysis of the exhaust gas has been used to indirectly estimate the biomass growth [38]. An important concept in the design of bioreactors is to allow a single vessel to have as many major operations as possible. The bioreactor is the core of the biological process with four major functions. They are: (1) to contain the substrate; (2) to contain the processed microorganism; (3) to protect the processed microorganism against contamination and (4) to control parameters and environmental conditions to optimise growth and product formation [39][40][41].
The most important factor need to be highlighted during microbial fermentation is sterilization.
According to Sindhu et al. [42], sterilization is mandatory to strictly control bio-contaminants. This was supported by Arora et al. [33] that SSF bioreactors must be completely sealed to prevent unwanted microorganisms or harmful environmental substances from entering into the bioreactor. Besides that, temperature, O2, CO2 and heat removal need to be carefully controlled and optimised [39]. The SSB design must consider design and operating variables, especially aeration transfer for effective diffusion of O2 in the gas phase, effective elimination of CO2 and metabolic heat, temperature control including the cooling system [33,34,39,43]. In SSF, O2 transfer can often be inefficient in some designs, whereby O2 transfer is ineffectively homogenously distributed into every particle of the solid substrate. Other difficulty would furthermore affect the temperature and water content of the solid medium, thus need to be tightly and concomitantly controlled and measured [16]. Constructive bioreactors have high biological reaction rates including microbial growth performance, substrate consumption, product formation and by-product synthesis rates [33,39,44]. Thus, it is very important to understand which of these criteria provides the best performance for bioreactors to be constructively designed. Yet, a greater understanding of the biological systems of microorganisms and biological processes in SSF are key to improve the design of bioreactors [32,45].
This work was carried out at the location where the bioreactor system was designed to generate moistened air. Here, two different arrangements were provided into the multi-layer tray SSB. The moistened air supplied water, O2 and facilitated heat and mass transfer to collectively improve fungal growth. In order to evaluate the performance of the bioreactor, final moisture content, O2 uptake rate (OUR), CO2 evolution rate (CER) and evolved temperature were investigated during fermentation. As analysis of O2 as well as CO2 in the exhaust gas is becoming generally accepted as a standard measuring technique in bioprocessing, the analysis was also applied in this study because it does not interfere with the sterile barrier. The results obtained will be discussed and reviewed for the two fungi reacting to every study design proposed.

Microorganisms and sporulation medium
The strains of Aspergillus awamori and Aspergillus oryzae were cultured and preserved in a solid sporulation medium containing 5% (w/v) whole wheat flour and 2% (w/v) agar (Sigma-Aldrich). The strains were activated in sterilised media and incubated over seven days at 32°C, then preserved at 4°C.
They were sub-cultured on an interval of every two months during the study.
Preparation of fungal spores as inoculum 0.5 mL spore suspension was transferred onto the surface of 100.0 mL of the same sporulation medium in a 500.0 mL Erlenmeyer flask and incubated for seven days at 32°C. After the incubation period, 50.0 mL of sterile 0.1% (v/v) Tween 80 solution and several sterile glass beads (4 mm diameter) were added to the flask. The spores were suspended by shaking the flask gently and then collected in a bottle as a spore suspension. Concentration of the spore suspension was measured by a haemocytometer. The volume of suspension needed for inoculation of the solid substrate was calculated for each experiment to reach a concentration of approximately 1.0 × 10 6 spores/g solid substrate.

Preparation of wheat bran as a solid substrate and inoculum transfer procedures
Wheat bran was obtained from the Cargill Wheat Processing Plant, Manchester, United Kingdom (UK).
The substrates were kept in an airtight container and stored in a cold room (4°C) for future use. Wheat bran is used without any treatment as a solid medium for growing A. awamori and A. oryzae.
Firstly, 20.0 g of wheat bran was weighed and placed into separate 250 mL flasks (in a total of eight flasks) before being sterilised at 121°C for 15 min. The substrates were allowed to cool at room temperature before inoculating with A. awamori and A. oryzae spores and being moistened with an amount of sterile distilled water to obtain the initial moisture content of 65%. About 1.2 × 10 6 spores/g substrate were inoculated into the flask and mixed well with a sterile spatula under aseptic conditions to uniformly distribute the spores within the substrate. After sufficient mixing, the content was distributed into each tray before being incubated at 32°C for 72 h. At the end of the fermentation period, a sample was taken for sample analysis.

Multi-layer squared tray solid state bioreactor
The bioreactor chamber consists of eight identical square trays perforated at the base and arranged in a The bioreactor chamber's assembly was configured as a module that fits in an autoclave. The bioreactor was autoclaved for 15 min at 121°C prior to operation. After cooling to room temperature, the reactor was filled with sterile inoculated substrate in a laminar flow cabinet under sterile conditions. Each tray can hold up to about 20.0 g of solid substrate with a 1.5 cm bed height. The tray medium enters the bioreactor chamber through the door. The door is then sealed to avoid any access from the environment into the system and vice versa. Each subsequent tray was assumed to be filled in the same thus are all under equal conditions. A schematic diagram of the experimental set-up consisting of the multi-layer tray SSB with aeration, control and on-line monitoring system is shown in Fig. 2.

Bioreactor setup and fermentation experiments
The bioreactor and solid substrate were autoclaved separately at 121°C for 15 min, prior to operation.
The sterilised substrate was inoculated with 1.2 x 10 6 spores/g substrate in another Erlenmeyer flask before loading into the sterile tray and before transfer into the bioreactor. All work was carried out in a laminar flow cabinet under sterile conditions. The bioreactor was then placed in an incubator room for 72 h at 32°C. Sterile moistened air was introduced as a fluidising medium at the bottom of and on the surface of the tray from the compressor depending on the air strategies applied to every experiment.
Two air flow rates were chosen as moistened air is sparged in two arrangements. Firstly, sterilised moistened air at a flow rate of 8 L/min was passed into an air distributor at the bottom of the bioreactor before being forced through the perforated mesh tray from bottom to the top (labelled as Exp 1).
Secondly, sterilised moistened air at a flow rate of 1 L/min was passed onto the surface of each fermented substrate (labelled as Exp 2). These air arrangements are described in Table 1. For moistened air, dry air from the compressor (Air compressor JUN-AIR, USA) was passed through a humidifier chamber before entering the system. The flow rate of the air into the bioreactor system was accurately measured and adjusted with a rotameter. The air was sterilised by passing it through a 0.45 µm cellulose acetate membrane filter and then into a humidifier chamber with sterilised distilled water which was monitored in a water bath at 30°C.

Measuring metabolic activity during solid state fermentation
Online monitoring of temperature Each tray was equipped with a thermocouple type K (Pico Lab Technology, UK) placed at the surface of the fermented substrate to continuously record the bed temperature over time. All of the on-line data were monitored on screen and recorded using a computer.

Online monitoring of respiratory gases
The system used for quantifying O2 and CO2 continuously measured exhaust gas compositions online.
An air outlet was situated in the headspace of the highest tray. The exit gas may need to be conditioned (moisture removed) before venting into the gas analyser. Therefore, the humid exhaust air from the bioreactor was firstly dried by passing through silica gel-containing tubes before entering an O2/CO2 analyser (FerMac 368 Electrolab, UK). This analyzer was connected to the Electrolab eLogger data logging and translates the concentration of O2 and CO2 to be recorded using a computer system.

Quantification of oxygen uptake and carbon dioxide evolution rates
In this study, O2 uptake rate (OUR) and CO2 evolution rate (CER) were calculated using the following equations [46].
O2 uptake rate (OUR): CO2 evolution rate (CER): Where; : This equation is based on the inert gas balance. It is assumed that CO2 is the only gaseous product of the fermentation process [46].

The Gompertz curve as a fungal growth curve
The Gompertz model was first initiated by Benjamin Gompertz (1825) and a thorough analysis of the model has been described by Winsor, (1932). The Gompertz model is a sigmoid function of the logistic curve and has been used as a growth curve, both for biological and economic phenomena [47]. An improvement to the model and derivation based on the systems theory was done by Skiadas and Skiadas [48]. In differential equation form, the model is based on this equation: Where; = the function of time = a positive constant expressing the rate of growth of the system Without loss of generality, the function of can be assumed bounded (0 < < 1, with = 1 corresponding to the entire population), in order that is the probability density's function of the growth process. Direct integration of equation 3 gives a solution to the Gompertz function [48]: The integrated Gompertz model, was used to analyze the kinetic data [49][50][51]. In this logistics-like model, the product ([ 2 ]) is a function of time ( ) according to the following equation. Where; : a constant related to the initial conditions (when = 0, then : the specific CO2 evolution rate (h -1 ) The constants [ 2 ], and were estimated from the data using a non-linear regression programme. The value of (h -1 ) refers to the time it takes for the value of [ 2 ] to reach [ 2 ].
Larger values of cause [ 2 ] to reach [ 2 ] in a shorter time. The value of (dimensionless) will determine the shape of the sigmoidal curve. Larger values of cause the initial exponential growth phase to be slower and the deceleration phase to reach asymptote faster. Smaller values of cause a rapid exponential growth phase and slower deceleration before the asymptote of [ 2 ] is reached [47]. Here, asymptote refers to a line that continually approaches a given curve but does not meet it in any finite distance.

Gas balance and calculation of respiratory quotient
The respiratory quotient (RQ) can be directly calculated when the composition of the exhaust gas is known according to the following equation: Where; : Respiratory quotient (dimensionless) : CO2 evolution rate (mole L -1 h -1 ) : O2 uptake rate (mole L -1 h -1 )

Determination of final moisture content
The moisture content of the samples was determined using the oven method, by measuring weight loss after heating to a constant weight at 95°C. Samples were placed in pre-dried metallic dishes with a known weight and immediately weighed. After being dried in the oven for 24 h, the samples were cooled for 30 min in desiccators before a final weighing.

Enzyme extraction and activity determination
After the 72 h fermentation period, samples were taken for enzyme (glucoamylase, protease, xylanase and cellulase) analysis. Fermented samples (2.0 g on a wet basis) were extracted with distilled water (40.0 mL) and agitated for 30 min on a rotary shaker (Infors A -CH 4103 Switzerland) at 250 rpm and 30 °C. Next, the solid suspensions were centrifuged at 10,000 rpm for 10 min (4 °C). The clear supernatant was used for enzyme analysis. Glucoamylase was assayed using a method as described by Ariff and Webb [52], using maltose as a substrate. Protease activity was measured using the ninhydrin colourimetric method as outlined by the European Brewery Convention with modifications made by Wang [53]. Determination of xylanase activity was conducted according to a method developed by Bailey et al. [54], while filter paper-based cellulase activity was measured according to IUPAC recommendations [55]. Here, filter paper Whatman No. 1 was employed as a substrate [56]. A standard operational procedure for enzymes analysis was developed in this study, as reported elsewhere [57].

Results and Discussion
Effect of air arrangements on final moisture content during fungal growth    [58]. Thus, in combination, wheat bran and A. oryzae were able to retain water and resulted in a high final moisture content in the fermented A. oryzae product.
This system was completely sealed with no access to the air from the inside or outside. However, large gas spaces in between the trays were included. In There are some key factors affecting the moisture content in SSF and it is important to know their influence on the processes. It could be summarised that A. awamori and A. oryzae were very versatile but also a sensitive type of fungi towards air arrangement in bioreactor systems. The use of moistened air as an alternative to provide adequate O2, is suitable for moisture content control. The air flow through the fermented substrate was also observed to play an important role in carrying moisture from the fermented substrate. When air was forced from the bottom to the top, it had a negative effect on A.
awamori culture but a positive effect on A. oryzae. When air was blown through the surface of the fermented substrate, it had a negative effect on the moisture content for both fungi. However, this is not the only factor to determine high productivity of the culture. At the initial fermentation stage, the temperature, O2 and moisture content are expected to be the same throughout the SSF system; however, as the fermentation progresses, O2 is consumed, heat is evolved and water is evaporated, and therefore gradients of temperature, moisture, gas, substrate and products are commonly observed in the fermented bed [14]. shows the fitting of the Gompertz model to these data for both fungi during SSF with wheat bran for the description of fungal growth. In this system, it is clear that air arrangement has a direct influence on the metabolic activity and evolved CO2. The strategy to supply air onto the surface of the fermented substrate (Exp 2) with moistened air at a flow rate of 1 L/min greatly enhanced the evolution of CO2 compared to Exp 1.

Gompertz curve and analysis of a distributed gas balance during fungal growth
For Exp 2 (for both A. awamori and A. oryzae), the evolution of CO2 was higher probably due to high concentration of O2 at the surface of fermented substrate. With a low flow rate at 1 L/min, the accumulation of O2 in the headspace (between the trays) was adequate to supply O2 for the fungi. Ultimately, given the assumptions to be considered, the Gompertz model shows potential. It has the ability to describe the varied outcomes under different culture conditions. Previously, the Gompertz model proved most suitable to fit the experimental data in a kinetic study on anaerobic treatment of a hazardous steel-mill waste known as rolling oil [59]. The biomass data obtained from the growth analysis of Streptomyces venezuelae for the effect of ultrasonication was well fitted to the Gompertz model [60]. Additionally, the Gompertz model predicted the microbial inactivation under varying time and temperature subjected to the surface of a food product. Here, the model required non-linear regression schemes and analyses were tested on pseudo-experimental data [61]. According to Kafle et al. [62], the modified Gompertz model fitted the experimental data better than a first-order kinetic model, during anaerobic treatment of apple waste with swine manure for biogas production. In addition, the modified Gompertz model best described the growth of Pseudomonas in raw pork under pallet packaging at all temperature conditions [63]. According to Augustine et al. [64], the Gompertz model does not incorporate the symmetry restriction, has a shorter period of fast growth and gave the best fit for growth kinetic study of filamentous fungi in SSF. suggested the use of the Gompertz model as a growth model and potential application to microcalorimetric data. Their study further observed the use of microcalorimetry in microbiology and biology, thus has become available to the end users of isothermal microcalorimetry. Instead of SSF, Augustine et al. [64] reported that the Gompertz model can be used to describe the growth of bacteria and yeasts in liquid media. Yet, the Gompertz and other log model possess similar properties that make them useful for the empirical representation of growth [47].

Quatification of OUR and CER to describe fungal growth
It is possible to estimate biomass development in SSF by considering OUR and CER, which are easily measurable parameters. OUR and and CER offer the advantage of a fast response time and are directly linked to the metabolism of the microorganism [38,70]. Fig. 5 shows OUR and CER profiles from four experiments carried out for both fungi.
The stationary growth phase for both fungi in all tray SSB systems were clearly reflected in the OUR and CER evolved heat curves. This growth phase was very short and lasted for 1 to 2 h of the fermentation time. The curves were not of a sigmoidal shape and reached the maximum value of O2 consumption, CO2 generation and heat evolution approximately in between 24 to 30 h of fermentation.
After this peak, the gradual decrease of heat and O2 and CO2 concentrations indicated the beginning of the last phase, which corresponds to slower fungal growth. This is a very slow phase of progress because the active spores still consume the O2 (plus other nutrients) and produce CO2 and heat. During the SSF of Rhizopus oligosporus with rice bran as the substrate, similarly, an increase in OUR and evolved heat at around 24 h of fermentation followed by a significance decrease was reported at 72 h [71]. In this scenario, it was considered that the high content of readily available starch, as a carbon source in wheat bran, was the reason for the comparatively high OUR, CER and evolved heat. This indicated high fungal growth during the first 24 to 30 h of SSF. Furthermore, in SSF, fungi cannot grow continuously as the amount of nutrients available is finite and waste products will accumulate. These conditions might be the reason for the deceleration of fungal growth after achieving maximum activity. Growth still continues but at a slower due to thepresence of nutrients.

Heat evolution as a mechanism to describe fungal growth
Temperature is the limiting design consideration for most SSB systems. A high amount of metabolic heat is produced during growth. The temperature of SSF cultures often increases to levels incompatible with growth. As the scale of SSB increases, the mechanism of heat transfer rapidly becomes insufficient.
The forced addition of air is usually necessary to allow evaporative cooling of the fermented substrate and also the system. Some form of moisture control is then required. Forced aeration with saturated moistened air can then be introduced into the system.
The effects of moistened air with two arrangements in multi-layer tray SSB are shown in Fig. 6 A better growth performance was observed in Exp 2 due to adequate O2 concentration. Furthermore, in Exp 2, the air was not forcefully blown through the bed but rather circulated through the headspace above the bed. Additionally, the substrate in the perforated mesh tray allowed better air circulation around the trays. Perforation in a tray's base allows air flow through the tray to assure uniform mass and heat transfer. The system was properly sealed with a gap of about 2.0 cm between the trays to allow better aeration and O2 accumulation.
In the current tray system of bioreactors, it was impossible to maintain the bed temperatures at the optimum value for growth, and therefore the fungus will suffer variations in temperature during the fermentation process. It is worth mentioning that from these studies, the amount of water loss and drying of the substrate were not drastic. Another important factor is the ability of the fungus to deal with fermentation conditions. For example, in this study, A. awamori might have suffered higher water loss compared to A. oryzae. However, the growth of A. awamori tended to be excellent in all experiments.
At the same time, inhibitory metabolites were increasing due to waste accumulation within the system.
The temperature often reaches values which severely limit growth or even kill the microorganims [8,19,65,70,71]. High temperatures might affect spore germination and growth, and product formation.
This can be observed in temperature profiles illustrated in Fig. 6 where a temperature of above 30°C were noted in all experiments.
The approach taken by creating moistened air flow in the system created a cooling system able to supply moisture into the fermentation substrate. At one stage, the substrate possessed enough moisture to support growth and fungal metabolic activities. The growth rate was expressed solely as a function of the current temperature according to the experimental results. The increment of temperature during SSF in the tray and the insulated packed-bed bioreactors was responsible for changes in the use of the soybean oil and in protease secretion by Yarrowia lipolytica [8]. A strategy that uses intermittent trickling of water in the bed bioreactor, results in good temperature control, avoiding the bed from drying, preventing substrate compaction, as well as increased Aspergillus ficuum mycelium growth and phytase production [13]. Evaluation of different airflow distribution techniques in a pilot-scale bioreactor by using an inner tube for air supply resulted in more homogenous enzyme production, with higher activities by the fungus, Myceliophthora thermophila I-1D3b [4].
Additionally, temperature can be expressed as a function of the evolved CO2 in the system. The profile for both parameters recorded in this work can almost provide an adequate estimation of the complete growth curve including the lag, log, exponential, and stationary growth phases. However, this profile cannot provide a complete representation that also includes the death phase. Instead, it clearly shows that the growth becomes slower after the exponential phase, as reported by Finkler et al. [72] on their investigation of heat and mass transfer done in the absence of growth. Furthermore, research on hydrodynamics under abiotic and biotic conditions in a novel bench-scaled wall-cooled tray SSB packed with moistened particles of agro-industrial waste, provided a focused understanding of the interaction with heat and mass transport mechanisms [73].

Respiration quotient and considerations about fungal growth
RQ simply explains the state of the microbial population in the fermentation process and gives an indication of the behaviour of metabolic activity. Accurate measurements of CO2 and O2, allow precise and instantaneous estimation of kinetic parameters associated with the respiration of the culture such as the specific OUR, CER and the RQ [38]. According to Equation 7, a theoretical RQ of the oxidation of simple sugars for aerobic microorganisms is equal to 1 [74,75].
( 6 12 6 ) + 6 2 → 6 2 + 6 2 The equation shows that during glucose fermentation through aerobic fermentation, the rate of O2 consumption is ideally six molecules, while six molecules of CO2 are produced per glucose molecule [74,75]. These deliberations are based on simple sugars, or related carbohydrates or polysaccharides [75]. It is important to know that solid substrate used in SSF has varying nutrients composition of carbon and nitrogen. Some contain complex compositions such as hemicellulose, cellulose and lignin. In this situation, microorganisms need to utilise the entire complex of the compounds and produce simple fermentative sugars before these can be easily consumed. In the practical terms in SSF, it is impossible to obtain the RQ equal to 1, but the probability of obtaining RQ < 1 and RQ > 1 is possible as shown in Equation 8.
When RQ < 1, it is assumed that one or more non-constitutive or newly induced metabolites have been possibly produced. CO2 was not the only product produced during the microbial fermentation.
According to Equation 8, other products, such as energy (ATP), water and secondary metabolites were produced. Yet, O2 is not only used for the respiration process but also for cell maintenance [74,75].
RQ > 1 indicates low O2 uptake and the occurrence of glucose fermentation. It is also indicates anaerobic fermentation. For example, in highly aerated fermentation, it is often considered that higher concentration of O2 was washed out, corresponds to badly functioning aeration and the existence of anaerobic zones in the fermenter system that must be immediately resolved [38,74,75].
Further discovery of off-gas analysis is provided in this work for multi-layer squared tray SSB with two varied air arrangements. The rates of O2 consumption and CO2 evolution are presented along with the calculated RQ values, giving further insights into the behaviour of the fermentation process. Fig. 7[a] shows the profiles of RQ obtained using Equation 6 for both fungi in multi-layer squared tray SSB with moistened air arrangements. The RQ values are presented in Fig. 7[b]. These RQ values were generated by obtaining the slope from every experiment illustrated in Fig. 7[a]. It was observed in these studies that all RQ values obtained were below 1 (Fig. 7[b]). Exp 1, the RQ value was 0.533 and 0.578 for A.
awamori and A. oryzae, respectively. Whilst, RQ values were higher in Exp 2. With the air arrangement in Exp 2, the RQ value for A. awamori and A. oryzae was 0.947 and 0.868, respectively. A. awamori showed RQ values around 1. The gas phase between the trays and the blowing of moistened air at 1 L/min onto the fermented surface provided a higher advantage for fungal growth. It was assumed that adequate O2 concentration was available in the system. This situation arises because of the perforations at the bottom of the trays, that facilitate mass transfer to the gaseous surrounding, both at the top and the bottom.
In SSF, the mycelium growth of fungi is restricted by the diffusion of O2 in the fermented solid substrate, especially in the inner depth [3]. Poor growth might occur deep within because O2 transfer inside the solid substrate is blocked due to the compact structure of the solid particles [3]. These situations were observed in rice koji at high moisture contents. Here, poor growth of the deeply submerged mycelium was probably caused by blocked O2 flow inside the rice grains [76]. The same observation was noted during the growth of A. niger in a tray SSF system that was limited by heat and O2 transfer, with the bed height of a tray reactor being limited due to heat, had mass transfer limitations and the presence of heat transfer gradients [77]. The study suggested that by increasing the thickness of the fungal biofilm around the solid particles, the porosity of the substrate bed and diffusivity of O2 in the bed were reduced.
According to Govind et al. [78], RQ values are close to 1 until O2 uptake reaches a maximum level and thereafter RQ becomes higher than 1 due to utilisation of metabolite products. Becerra and Gonzalez-Siso [79] argued that changes in RQ values could be associated with the production of multiple products by Streptomyces cattleya during SSF. Barrios-Gonzalez et al. [80] reported that RQ of 0.7 obtained from their work indicated oil-supported metabolism for Nocardia lactamdurans during the production of efrotomycin in SSF. As recommended practise based on a simulation work by Rodríguez-León et al. [75], RQ patterns indicated the kind of kinetics involved in a particular fermentation process influenced by metabolite productions and microbial growth that can be categorized as (1) associated growth (RQ value close to 1); (2) partially associated growth (RQ value lower than 1 in the case the substrates are polysaccharides from simple sugars units) and (3) non-associated growth (RQ value lower than 1 in the case sugar or related molecules are the substrate) [75]. Overall, the RQ values must be determined for particular processes and conditions [38].
The correlation between RQ and production of CO2 during fungal growth consumption and CO2 evolution rate [38,75]. Thus, it would be convenient for a basic laboratory SSF bioreactor to have a gas analysis system at the bioreactor's outlet. A simulation work by Mann et al. [81], described the calculation of the ratio of evolved CO2 similar to the RQ for aerobic fermentation, allows the prediction of whether acetate or ethanol is predominantly produced during syngas fermentation in quasi-continuously ventilated shake flasks using Clostridium ljungdahlii. Different types of solid substrates, microorganisms, SFF bioreactor systems and the overall metabolic pathway must be taken into account to obtain a proper RQ value [34,74,75]. Mendez-Gonzalez et al. [34] reported that conidia production and productivity were significantly enhanced with packed bed column with forced aeration compared to tray and plastic bag bioreactors. Forced aeration allowed online measurement of CO2 production and proved to have an important role in determining when to harvest conidia from Metarhizium robertsii in SSF processes.

The correlation between heat evolution with CER during fungal growth
One of the most remarkable observational findings was the strong correspondence between the increase of temperature and the concentration of evolved CO2 during the 72 h fermentation. A high degree of correlation was found to relate the evolving CO2 and metabolic heat, as a result of carbon utilization.
During the period of CO2 evolution, the total amount of evolved CO2 with time was found to correlate with changes in evolved temperature during microbial activity. Meanwhile, during respiration, a process dependent on O2 consumption and evolved CO2, was observed to be highly exothermic. Furthermore, heat generation was directly related to the level of metabolic activities of the microorganisms [3].
Motivated by these observations, evaluation of CER data and temperature changes during SSF for both fungi were carried out. Fig. 9 shows the profiles of temperature changes and CER over time for A. awamori and A. oryzae. Heat and CO2 are microbially produced, thus indicate the existence of microbial metabolic activity, consumption of O2, evolution of CO2 and utilization of organic carbon [82]. In the case of aerobic microorganisms, the catabolic heat evolution rate is regarded to be proportional to the OUR and CER.
According to Cooney et al. [83], if the heat evolution rate transfer ratio is constant, the change of temperature can be calculated by monitoring O2 consumption or CO2 evolution. The rising CO2 levels correlated with rising temperatures because CO2 is believed to trap natural heat from the surface warming of fermented substrate [82]. The study further elaborated that CO2 would enclose the surface and trap heat, causing the fermented bed surface to become insulated and ultimately causing a temperature rise. In addition, the evolution of heat during microbial activity can provide useful data, such as growth rate, lag phase, or maximum growth [69]. In addition, encouragingly, the use of this technique in the biological and biomedical field has been increasing. Examples include detection and characterization of pathogens, drug testing, parasitology and tissue engineering [69].

Effect of air arrangement on spore and enzyme production
An average value of spore production and levels of enzymatic activities resulted from the moistened air arrangement with different flow rate are shown in Fig. 10 and Fig. 11, respectively. From these observations, A. awamori is an excellent fungus for spores and glucoamylase, xylanase and cellulase production. However, A. awamori is a poor protease producer. A. oryzae is excellent in protease production but poor in glucoamylase, xylanase and cellulase production. This might be because A.
oryzae seemed to favour a solid substrate with high protein content (as a nitrogen source) such as rapeseed meal, and does not favour wheat bran as a main substrate [57]. A. awamori seemed to favour a solid substrate with a high carbohydrate content (as a carbon source) [57].
In all experiments carried for both fungi, moistened air at a flow rate of 1 L/min (Exp 2) favour spores and enzymes production. This strategy was believed to create homogenous conditions in term of an enough supply of O2 concentration while maintaining moisture contents and avoiding bed drying. In Exp 1, a high flow rate of 8 L/min tend to create stress in the bioreactor's system, whereby most O2 was "washed" out and concentrations of O2 might be low. A high flow rate also gave some negative effects on the moisture content, especially for A. awamori (Fig. 3). We conclude that with proper strategy to supply air into the bioreactor system for SSF, is advantageous to the fermentation's performance and fungal activity. The strategy suggests that reasonable control of the O2 concentration, bed temperature and moisture content can be achieved by manipulating the flow rate of moistened air. As reported by Perez et al. [4,5], using an inner tube for air supply resulted in more homogeneous enzyme production, with higher activities and solving overheating and gaseous exchanges. However, our results contrast with previous reports by Castro et al., 2015 [84]. In this study SSF in SSB with forced aeration showed high levels of a cocktail of hydrolases (amylases, cellulases, xylanases and proteases) by A. awamori.
This was supported by Mendez-Gonzlez et al. [34] which showed that forced aeration during SSF in SSB allows high conidia production and productivity. However, for a better monitoring and good comparison, other solid substrates, different types of microorganisms or fungi and different types of bioreactors, should be investigated.

Conclusion
This work presented two different moistened aeration strategies in newly-designed multi-layer eight squared tray SSB containing wheat bran to cultivate the fungus A. awamori and A. oryzae by solid state fermentation. Experiments were carried out under sterile conditions, equipped with an online gas analyse and temperature recorder, for continuous O2, CO2 and metabolic heat measurement. Continuous on-line monitoring of fungal growth could be performed by indirect methods, including monitoring O 2 consumption, CO 2 and heat evolution during SSF, is very useful to assess the physiological state and respiration rate of culture. Yet, it can directly reveal the activity of the A. awamori and A. oryzae culture in SSF and can be observed as indirect techniques for biomass estimation. Reasonable measure of the final moisture content can be achieved through strategy with moistened air at inlet with a flow rate 8 L/min and 1L/min. The air flow distribution strategies and the bioreactor with multi-layered trays contributes to an effective air distribution and optimal temperature maintenance. Even though, the final moisture content and temperature profiles for both fungi were little affected by the alternative of airflow distribution, one might conclude that the introduction of moistened air produced a better distribution of gas within the bioreactor. CO 2 that evolved as a result of metabolic activity during A. awamori and A.
oryzae SSF on wheat bran is easy to handle with a simple Gompertz model and fit the integrated CO 2 data and predict the quantity of CO 2 evolution in all experiments. Measurement of OUR and CER will lead to determination of RQ that gave important information about the fungal growth. In all respective experiments carried out in this work, the RQ values obtained are less than 1. Yet, a strategy with different aeration arrangements improved productivity of fungi, including production of spores and consortium of enzymes (glucoamylase, protease, xylanase and cellulase).
Even though the present study is on a small scale, it is hoped that the multi-layer squared tray SSB systems will provide a greater and general understanding information when looked at in conjunction with other studies in open literature. This work suggests that there will be a remarkable improvement in bioreactor productivity and better fungal growth. Information obtained could have several potential advantages including, contributing to future designs and applications. Some more efforts must be done to improve the performance of the bioreactor; for example further investigations of mass transfer and possibly to scale-up into larger bioreactor by solving problems of overheating and gaseous exchanges. Figure 1 A schematic of a multi-layer tray SSB viewed from the side, with emphasis on the location of the tray  Fermentation summary of average enzymes production from multi-layer tray SSB with two air arrangements.

Figures
[AA] -A. awamori and [AO] -A. oryzae. The results are shown as an average from eight trays and mean ± standard deviation

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